Francisella tularensis, the bacterial cause of tularemia, is a highly virulent intracellular pathogen and a Tier 1 select agent because of its potential to cause severe disease as an airborne bioweapon. The virulence of F. tularensis is linked to its capacity to evade recognition by the host and actively suppress innate immune responses. Because specialized facilities are required for work with fully virulent F. tularensis (Ft), much of the research in this field has been done with closely related but less virulent strains of Francisella, including Francisella novicida (Fn), which is highly virulent for mice but not for humans. Pneumonia is the most lethal form of tularemia, and the target cell of Francisella in the lungs is the alveolar macrophage (AM). The interaction of F. tularensis with human AM has not been studied. The overall goal of this project is to identify key mechanisms underlying the parasitism of human AM, using an approach that compares the interactions of human and murine AM with Ft, virulent for both mice and humans and with Fn, virulent only for mice. Specific Aim #1. Determine if F. tularensis evades and/or suppresses the immune responses of human and murine AM. This aim will test the hypothesis that virulent Ft parasitizes human AM by both evading recognition and actively suppressing innate immune responses. The approach will be to: 1) Compare the replication of Ft and Fn in human and murine AM; 2) Develop comparative transcriptional profiles of the responses of human and murine AM to infection with Ft and Fn; 3) Measure the release of cytokines that are likely to be important in the host response to Ft: TNF-?, IL-12/23 p40, IL-10, TGF-?, IFN-?, and IFN-?; 4) Determine if Ft or Fn infection inhibits responsiveness to secondary stimulation with Toll-like receptor ligands; and 5) Determine if Ft or Fn infection inhibits responsiveness to IFN-?. Specific Aim #2. Determine if F. tularensis induces or suppresses inflammasome activation in human and murine AM. This aim will test the hypothesis that virulent Ft fails to activate the inflammasome in human or murine AM, thereby stifling the production of IL-1? and IL-18, and preserving its intracellular niche by preventing pyroptosis. The approach will be to: 1) Determine if Ft and Fn activate the inflammasome in human and murine AM by measuring caspase-1 activation, release of IL-1? and IL-18, and pyroptosis; 2) Determine if Ftt or Fn suppresses inflammasome activation by exogenous triggers; 3) Determine if Ftt or Fn LPS trigger non-canonical inflammasome activation; and 4) Determine if inflammasome activation induces a protective response to Ft and whether this protection is mediated by IL-1?/IL-18 production or pyroptosis. These studies will yield novel insights into the interaction of F. tularensis with human AM that will guide subsequent work directed at further understanding of the mechanisms underlying the pathogenesis of pneumonic tularemia, informing the development of novel strategies for therapeutic intervention.